Isolated GH deficiency (IGHD) is familial in up to 30% of the cases. The most common form (type IB) has autosomal recessive transmission. Two mutations (generating truncated receptor) of the GHRH receptor (GHRHR) gene were first described as the basis for IGHD IB in two large kindreds. We have established the molecular heterogeneity of this disease through identification of ten additional mutations of the GHRHR, proving that defects in the GHRHR are common in familial IGHD lB. They consist of a promoter defect, one splice mutation, one nonsense mutation, one deletion, and six missense mutations that replace conservative amino acids: H137L, L144H, A176V, A222E, F242C, K329E. Preliminary characterization of the missense mutant receptors expressed in CHO cells shows that all these mutant receptors fail to produce a normal a cAMP response after exposure to GHRH, confirming that they are mutations and not innocent polymorphisms. Further characterization will reveal important information about receptor domains and specific amino acids that control receptor interaction with GHRH and with G proteins, and receptor desensitization. Specific aim 1: We have created cDNA's encoding for each of the mutant GHRHR's containing GFP or FLAG tag. We will express these fusion receptors in mammalian cells to determine the basis for their defective signaling. If proper cell surface expression is confirmed, binding studies will determine if the mutant receptors are able to bind their ligand. If the mutations cause abnormal cell surface expression, further experiments will determine the mechanism of such lack of expression. The wild type and mutant receptors will also allow us to study if and how specific amino acid substitutions interfere with receptor interaction with Gs alpha and other downstream proteins. Specific aim 2: We propose to study if the GHRHR exists as dimer using the novel BRET technique, if dimerization is ligand-dependent, and if it is prevented by any of the six the mutations. Altogether, these experiments will teach us new information about the pathophysiology of GHRHR signaling in health and disease. [unreadable] [unreadable]